| Summary: | 碩士 === 台北醫學院 === 醫學研究所 === 85 === Fibroblast-collagenase (collagenase-1, matrix
metalloproteinase-1, MMP-1) is an unique enzyme for the initial
cleavage of interstitial collagen types I and III. It is thought
to play a major role in the degradation of extracellular matrix
during the pathogenesis of many important human diseases such as
cancer, rheumatoid arthritis, and periodontitis. Our previous
studies applied with enzymatic hydrolysis of rat tail tendon
collagen, Western blotting and others have proved that TPA-
treated normal human keratinocytes (NHKs) de novo synthesis and
secret procollagenase/collagenase-1 in culture. Studies with
indirect immunofluorescent technique have also demonstrated that
the cultured NHKs with TPA treatment produce procollagenase/
collagenase-1 mainly distributing in cytoplasm especially around
perinuclear region, but definite localization could not be
determined by this immunofluorescent technique. The purpose of
the present research is to find the intracellular localization
of synthesized procollagenase and its possible secretory
pathway. For this, immunoelectron microscopic studies in the
ultrastreptoavidin-biotin method to localize procollagenase/
collagenase intracellularly were performed in cultured NHKs with
or without TPA treatment. In the preliminary research, the
cultured NHKs without immunolabeling were studied in the
transmission electron microscopy. The cultured NHKs consisted of
heterogeneous subpopulations morphologically. The smaller and
less differentiated cells contain small villous projections on
the surfaces, while the larger ones contain broad and irregular
processes. The Golgi apparatus, rough endoplasmic reticulum and
multivescular bodies of the smaller cells are less prominent
than those of the larger ones. The tonofilaments of cytokeratins
are more abundant in the larger ones. Then immunoelectron
microscopic study was performed and showed that procollagenase/
collagenase were located in rough endoplasmic reticulum, Golgi
apparatus and outer surface of cell membrane. The pattern of
subcellular localization in cultured NHKs was the same as that
in the synovial fibroblasts. The immunolocalization of
procollagenase /collagenase in the outer surface of cell
membrane suggested that procollagenase/collagenase may be
involved in the proteinases cascade around the pericellular
space of NHKs.
|
|---|
